A rapid in situ procedure for determination of bacterial susceptibility or resistance to antibiotics that inhibit peptidoglycan biosynthesis

BACKGROUND: Antibiotics which inhibit bacterial peptidoglycan biosynthesis are the most widely used in current clinical practice. Nevertheless, resistant strains increase dramatically, with serious economic impact and effects on public health, and are responsible for thousands of deaths each year. C...

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Autores: Santiso Brandariz, Rebeca, Tamayo Novas, María, Gosálvez, Jaime, Bou Arévalo, Germán, Fernández López, María Carmen, Fernández García, José Luis
Tipo de recurso: artículo
Fecha de publicación:2011
País:España
Institución:Servizo Galego de Saúde (SERGAS)
Repositorio:RUNA. Repositorio da Consellería de Sanidade e Sergas
OAI Identifier:oai:runa.sergas.gal:20.500.11940/3137
Acceso en línea:http://hdl.handle.net/20.500.11940/3137
Access Level:acceso abierto
Palabra clave:Anti-Bacterial Agents
Bacteria
Bacterial Infections
Down-Regulation
Drug Resistance, Bacterial
Microbial Sensitivity Tests
Peptidoglycan
Antibacterianos
Bacterias
Infecciones Bacterianas
Regulación hacia Abajo
Farmacorresistencia Bacteriana
Pruebas de Sensibilidad Microbiana
Peptidoglicano
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spelling A rapid in situ procedure for determination of bacterial susceptibility or resistance to antibiotics that inhibit peptidoglycan biosynthesisSantiso Brandariz, RebecaTamayo Novas, MaríaGosálvez, JaimeBou Arévalo, GermánFernández López, María CarmenFernández García, José LuisAnti-Bacterial AgentsBacteriaBacterial InfectionsDown-RegulationDrug Resistance, BacterialMicrobial Sensitivity TestsPeptidoglycanAntibacterianosBacteriasInfecciones BacterianasRegulación hacia AbajoFarmacorresistencia BacterianaPruebas de Sensibilidad MicrobianaPeptidoglicanoBACKGROUND: Antibiotics which inhibit bacterial peptidoglycan biosynthesis are the most widely used in current clinical practice. Nevertheless, resistant strains increase dramatically, with serious economic impact and effects on public health, and are responsible for thousands of deaths each year. Critical clinical situations should benefit from a rapid procedure to evaluate the sensitivity or resistance to antibiotics that act at the cell wall. We have adapted a kit for rapid determination of bacterial DNA fragmentation, to assess cell wall integrity. RESULTS: Cells incubated with the antibiotic were embedded in an agarose microgel on a slide, incubated in an adapted lysis buffer, stained with a DNA fluorochrome, SYBR Gold and observed under fluorescence microscopy. The lysis affects the cells differentially, depending on the integrity of the wall. If the bacterium is susceptible to the antibiotic, the weakened cell wall is affected by the lysing solution so the nucleoid of DNA contained inside the bacterium is released and spread. Alternatively, if the bacterium is resistant to the antibiotic, it is practically unaffected by the lysis solution and does not liberate the nucleoid, retaining its normal morphological appearance. In an initial approach, the procedure accurately discriminates susceptible, intermediate and resistant strains of Escherichia coli to amoxicillin/clavulanic acid. When the bacteria came from an exponentially growing liquid culture, the effect on the cell wall of the beta-lactam was evident much earlier that when they came from an agar plate. A dose-response experiment with an E. coli strain susceptible to ampicillin demonstrated a weak effect before the MIC dose. The cell wall damage was not homogenous among the different cells, but the level of damage increased as dose increased with a predominant degree of effect for each dose. A microgranular-fibrilar extracellular background was evident in gram-negative susceptible strains after beta-lactam treatment. This material was digested by DNase I, hybridised with a specific whole genome probe, and so recognized as DNA fragments released by the bacteria. Finally, 46 clinical strains from eight gram-negative and four gram-positive species were evaluated blind for susceptibility or resistance to one of four different beta-lactams and vancomycin, confirming the applicability of the methodology. CONCLUSION: The technique to assess cell wall integrity appears to be a rapid and simple procedure to identify resistant and susceptible strains to antibiotics that interfere with peptidoglycan biosynthesis.2011info:eu-repo/semantics/articlehttp://hdl.handle.net/20.500.11940/3137reponame:RUNA. Repositorio da Consellería de Sanidade e Sergasinstname:Servizo Galego de Saúde (SERGAS)Ingléshttp://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessoai:runa.sergas.gal:20.500.11940/31372026-06-12T08:40:47Z
dc.title.none.fl_str_mv A rapid in situ procedure for determination of bacterial susceptibility or resistance to antibiotics that inhibit peptidoglycan biosynthesis
title A rapid in situ procedure for determination of bacterial susceptibility or resistance to antibiotics that inhibit peptidoglycan biosynthesis
spellingShingle A rapid in situ procedure for determination of bacterial susceptibility or resistance to antibiotics that inhibit peptidoglycan biosynthesis
Santiso Brandariz, Rebeca
Anti-Bacterial Agents
Bacteria
Bacterial Infections
Down-Regulation
Drug Resistance, Bacterial
Microbial Sensitivity Tests
Peptidoglycan
Antibacterianos
Bacterias
Infecciones Bacterianas
Regulación hacia Abajo
Farmacorresistencia Bacteriana
Pruebas de Sensibilidad Microbiana
Peptidoglicano
title_short A rapid in situ procedure for determination of bacterial susceptibility or resistance to antibiotics that inhibit peptidoglycan biosynthesis
title_full A rapid in situ procedure for determination of bacterial susceptibility or resistance to antibiotics that inhibit peptidoglycan biosynthesis
title_fullStr A rapid in situ procedure for determination of bacterial susceptibility or resistance to antibiotics that inhibit peptidoglycan biosynthesis
title_full_unstemmed A rapid in situ procedure for determination of bacterial susceptibility or resistance to antibiotics that inhibit peptidoglycan biosynthesis
title_sort A rapid in situ procedure for determination of bacterial susceptibility or resistance to antibiotics that inhibit peptidoglycan biosynthesis
dc.creator.none.fl_str_mv Santiso Brandariz, Rebeca
Tamayo Novas, María
Gosálvez, Jaime
Bou Arévalo, Germán
Fernández López, María Carmen
Fernández García, José Luis
author Santiso Brandariz, Rebeca
author_facet Santiso Brandariz, Rebeca
Tamayo Novas, María
Gosálvez, Jaime
Bou Arévalo, Germán
Fernández López, María Carmen
Fernández García, José Luis
author_role author
author2 Tamayo Novas, María
Gosálvez, Jaime
Bou Arévalo, Germán
Fernández López, María Carmen
Fernández García, José Luis
author2_role author
author
author
author
author
dc.subject.none.fl_str_mv Anti-Bacterial Agents
Bacteria
Bacterial Infections
Down-Regulation
Drug Resistance, Bacterial
Microbial Sensitivity Tests
Peptidoglycan
Antibacterianos
Bacterias
Infecciones Bacterianas
Regulación hacia Abajo
Farmacorresistencia Bacteriana
Pruebas de Sensibilidad Microbiana
Peptidoglicano
topic Anti-Bacterial Agents
Bacteria
Bacterial Infections
Down-Regulation
Drug Resistance, Bacterial
Microbial Sensitivity Tests
Peptidoglycan
Antibacterianos
Bacterias
Infecciones Bacterianas
Regulación hacia Abajo
Farmacorresistencia Bacteriana
Pruebas de Sensibilidad Microbiana
Peptidoglicano
description BACKGROUND: Antibiotics which inhibit bacterial peptidoglycan biosynthesis are the most widely used in current clinical practice. Nevertheless, resistant strains increase dramatically, with serious economic impact and effects on public health, and are responsible for thousands of deaths each year. Critical clinical situations should benefit from a rapid procedure to evaluate the sensitivity or resistance to antibiotics that act at the cell wall. We have adapted a kit for rapid determination of bacterial DNA fragmentation, to assess cell wall integrity. RESULTS: Cells incubated with the antibiotic were embedded in an agarose microgel on a slide, incubated in an adapted lysis buffer, stained with a DNA fluorochrome, SYBR Gold and observed under fluorescence microscopy. The lysis affects the cells differentially, depending on the integrity of the wall. If the bacterium is susceptible to the antibiotic, the weakened cell wall is affected by the lysing solution so the nucleoid of DNA contained inside the bacterium is released and spread. Alternatively, if the bacterium is resistant to the antibiotic, it is practically unaffected by the lysis solution and does not liberate the nucleoid, retaining its normal morphological appearance. In an initial approach, the procedure accurately discriminates susceptible, intermediate and resistant strains of Escherichia coli to amoxicillin/clavulanic acid. When the bacteria came from an exponentially growing liquid culture, the effect on the cell wall of the beta-lactam was evident much earlier that when they came from an agar plate. A dose-response experiment with an E. coli strain susceptible to ampicillin demonstrated a weak effect before the MIC dose. The cell wall damage was not homogenous among the different cells, but the level of damage increased as dose increased with a predominant degree of effect for each dose. A microgranular-fibrilar extracellular background was evident in gram-negative susceptible strains after beta-lactam treatment. This material was digested by DNase I, hybridised with a specific whole genome probe, and so recognized as DNA fragments released by the bacteria. Finally, 46 clinical strains from eight gram-negative and four gram-positive species were evaluated blind for susceptibility or resistance to one of four different beta-lactams and vancomycin, confirming the applicability of the methodology. CONCLUSION: The technique to assess cell wall integrity appears to be a rapid and simple procedure to identify resistant and susceptible strains to antibiotics that interfere with peptidoglycan biosynthesis.
publishDate 2011
dc.date.none.fl_str_mv 2011
dc.type.none.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv http://hdl.handle.net/20.500.11940/3137
url http://hdl.handle.net/20.500.11940/3137
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.rights.none.fl_str_mv http://creativecommons.org/licenses/by/4.0/
info:eu-repo/semantics/openAccess
rights_invalid_str_mv http://creativecommons.org/licenses/by/4.0/
eu_rights_str_mv openAccess
dc.source.none.fl_str_mv reponame:RUNA. Repositorio da Consellería de Sanidade e Sergas
instname:Servizo Galego de Saúde (SERGAS)
instname_str Servizo Galego de Saúde (SERGAS)
reponame_str RUNA. Repositorio da Consellería de Sanidade e Sergas
collection RUNA. Repositorio da Consellería de Sanidade e Sergas
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15,301603

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